Field of the Invention
The invention broadly relates to electrochemical biosensing, more specifically to an electrochemical biosensing device to detect the activity of proteases and active infection in biological fluids and tissues, and even more particularly to a method of electrochemical biosensing to detect the activity of proteases and active infection in biological fluids and tissues.
Description of the Related Art
Differentiated from acute wounds that heal in a matter of days or weeks, chronic wounds persist for months or years and, may, last a lifetime. Such chronic wounds are commonly referred to as non-healing wounds. Examples include diabetic foot ulcers, venous leg ulcers, and pressure ulcers, and other types of chronic wounds including arterial ulcers, non-healing surgical wounds, burns, fistulae, dermatitis or vasculitis wounds, skin cancers, and radiation wounds. Unlike acute wounds that progress through the four normal phases of healing (hemostasis, inflammation, proliferation, and remodeling), chronic wounds stall in their healing, often in the inflammatory phase. Wounds fail to heal because molecular and cellular abnormalities in the wound prevent healing progress.
Over the past decade, expert panels have met at international wound meetings to report the importance of effective assessment and diagnosis in the treatment of wounds. The first such international group of experts met in June, 2007, with the intent that their report would serve as a platform to stimulate the development of dedicated test kits that would beneficially influence the future management of problem wounds. This panel concluded that specific biochemical tests were urgently needed that would identify the causes of delayed healing in wounds that are slow-to-heal and that the most appropriate markers to serve as biochemical indicators of stalled wound healing would include elevated proteases, infection, and perhaps decreased nitric oxide.
A second panel of experts of international recognition convened in February, 2011, and confirmed the need for biochemical wound diagnostics and identified elevated wound proteases as an important biochemical target. This panel also determined that an easy to use, point-of-care protease diagnostic would be of immense value in clinical practice. A third expert panel also convened in May, 2011, and concluded that wound protease levels would be a useful marker to help direct clinicians to specific treatments. The proteases most often elevated in non-healing wounds that are often stalled in an inflammatory state include matrix metalloproteinases, especially collagenases and gelatinases, and serine proteases, especially neutrophil serine proteases. The literature indicates that multiple matrix metalloproteinases (MMPs) are associated with stalled wound healing. These proteases are normal contributors to the early inflammatory phase of wound healing, to degrade damaged tissue and help prepare a clean wound bed for healing. But this activity normally occurs within three to five days after which the activities of these proteases reduce to very low levels in the wound environment. However, in non-healing wounds elevated levels of proteases persist and they continue to degrade extracellular matrix, growth factors and receptors involved in wound healing. This causes the wound not to fully heal.
Multiple matrix metalloproteinases contribute to stalled wound healing including the gelatinases, MMP-2, and MMP-9; the collagenases MMP-1, MMP-8, and MMP-13; stromelysin, MMP-3; and matrilysin, MMP-7. Whereas some researchers tried to identify one of these to provide the best target for diagnostic test development, other researchers have proposed that the measurement of just one individual protease would be unlikely to be representative of the proteolytic environment of the wound. Although there have now been 25 MMPs described and classified, partly on historical assessment of the substrate specificity of the MMP (and partly on the cellular localization of each), it has become increasingly clear that there is a lot of overlap in their substrate specificities. This is not surprising since structural analysis of the catalytic domains of many MMPs revealed that their polypeptide folds are essentially superimposable. Furthermore, it has been shown that substrate specificities that might exist in their natural substrates further break down when short peptide substrates are used, due to the loss of secondary and tertiary structure that only exists in the full-length natural protein substrates. Peptide substrates have been identified that can be cleaved by multiple wound MMPs, and these promiscuous substrates would make appropriate reagents for a more representative determination of the total proteolytic activity in the wound environment.
In addition to the presence of elevated MMP proteases in a wound, a large body of work has also identified that infection of wounds is also a major factor that prevents them from healing. Common wound pathogens include, but are not limited to, Staphylococcus aureus (including MRSA) and Pseudomonas aeruginosa. These bacteria exist in two major forms: either free-living single cells known as planktonic form, or as biofilm communities. Whereas planktonic bacteria are relatively easy to control (unless they consist of superbugs such as MRSA [Methicillin-resistant Staphylococcus aureus], C. Diff [Clostridium difficile], VRE [Vancomycin-resistant Enterococci], CRE [Carbapenem-resistant Enterobacteriaceae] and CRKP [Carbapenem-resistant Klebsiella pneumonia]), bacterial biofilms are much more difficult to remove.
A biofilm is an assemblage of microbial cells that is irreversibly associated with a surface and is most often enclosed in a matrix of primary polysaccharide material often referred to as ‘slough’ in chronic wounds. Biofilm-associated organisms differ from their planktonic counterparts in the genes that are transcribed, resulting in an altered resistance to antibiotics and the human immune system. A biofilm colony is more similar to a multicellular organism with cell-cell interaction and communication and division of labor, and this makes such colonies very difficult to kill and control. In a biofilm colony there are some bacteria that are growing and reproducing and making the matrix they live in, while other cells exist in a protective, sprawling state that are relatively quiescent, but in the event of a catastrophic challenge, those cells can survive and re-seed the biofilm. For example, biofilm can often reestablish within twenty four (24) hours following removal by surgical debridement.
Biofilm in wounds has become a significant problem. It has been estimated that up to sixty percent (≤60%) of non-healing wounds contain biofilm but it is difficult to diagnose. Some researchers suggest measuring wounds for the presence of common wound bacteria, but others point out that just as they are present on normal skin surfaces all over the human body, commensal bacteria also exist on wound surfaces that are not causing active infection. A diagnostic that cannot distinguish between an active infection and a passive microbial colonization is of little use as an indicator of the state of the wound with respect to whether the presence of microbes is preventing the wound from healing.
As used herein, an ‘active’ infection is an infection caused by microorganisms, whether planktonic or associated in biofilm communities, that are maintaining a chronic state of wound inflammation or contributing to wound deterioration and preventing wound healing. Other suggestions for detecting the presence of biofilm include measuring components of the biofilm matrix, including polysaccharide, proteinaceous, or nucleic acid components. Such measurements, however, miss active infection caused by planktonic bacteria, and there are other problems with using such targets.
There is a need for an invention that recognizes that a better indicator of active infection than bacteria or their constituents is a measurement of the stressful response of the host to the presence of active infection. In response to a microbial infection, the host mounts a rapid multi-faceted immune response. For the purposes of the present invention, this includes the sending of neutrophil granulocytes, which are an abundant type of white blood cells adept at killing and phagocytosing microorganisms and other foreign material. Neutrophils (also known as leukocytes) are one of the first responders of inflammatory cells to migrate towards sites of inflammation. They migrate through the blood vessels, then through interstitial tissue, following chemical signals from pro-inflammatory cytokines such as Interleukin-1 (IL-1), IL-8, and tumor necrosis factor-alpha (TNF-α), in a process called chemotaxis. While some have suggested the detection of the pro-inflammatory cytokines as suitable targets for the diagnosis of infection, there is still a need to measure the activity of host proteases as a more informative analyte. This is because the host proteases released from neutrophils are closest to describing the effects of infection because they are the specific entities performing the tissue damage. In addition, the concentrations of cytokines are much lower and therefore more difficult to detect and interpret.
When neutrophils encounter a bacterial cell or foreign body, they become activated where they first initiate an oxidative attack against the bacterial cell wall and then release granules from their cytoplasm containing high concentrations of proteases into the bacteria through the hole in its outer cell membrane caused by the oxidative burst. These neutrophil proteases lead to the death of the pathogen by breaking down critical proteins inside the bacteria. Neutrophils also recognize biofilm as foreign and mount their attack against it, but the bacteria can ‘hide’ within the biofilm matrix, inaccessible to and protected from both the oxidative burst and the neutrophil proteases. The chronic presence of the biofilm colony causes the host's immune system to continually send neutrophils to try to remove it, resulting in high local concentrations of proteases in the wound, released from the activated neutrophils, and these cause significant damage to host tissue.
This process occurs in non-healing (chronic) wounds causing degradation of newly developing extracellular matrix, but it also occurs in other host tissues resulting in multiple disease states including, but not limited to, chronic obstructive pulmonary disease, COPD, including chronic bronchitis and emphysema, and periodontitis (destruction of gum tissue). The most notable proteases contributing to this tissue damage by the host's own attack response are the neutrophil serine proteases (NSPs) secreted from neutrophil azurophilic granules including neutrophil elastase, proteinase-3, and cathepsin G.
Accordingly, there is a long-felt need for a device and method for detecting the activity of proteases and active infection in biological fluids and tissues.